Aspartic proteases are peptidases present in animals, plants, fungi and viruses and exhibit a wide range of functions and activities, including: mammalian digestion e.g. chymosin and pepsin A, activation and degradation of polypeptide hormones and growth factors e.g. cathepsin D, regulation of blood pressure e.g. rennin, degradation of haemoglobin by parasites e.g. plasmepsins, proteolytic processing of the HIV polyprotein e.g. retropepsin, involvement in pollen-pistil interactions in plants e.g. Cardosin A.
Aspartic proteases are synthesised as preproenzymes and contain a signal peptide, which is cleaved resulting in a proenzyme which can be secreted and activated autocatalytically. Generally, aspartic proteases consist of a single peptide chain of approximately 320-360 amino acid residues, composed mainly of n-strand structures arranged into two lobes. The catalytic site of the enzyme is located between these two lobes, each containing an aspartate residue which are within hydrogen-bonding distance of each other and act together to activate a water molecule which results in cleavage of the substrate peptide bond (via nucleophilic attack).
Plant aspartic proteases differ from other aspartic proteases in that they comprise a Plant Specific Insert which is cleaved out during protein maturation, besides a signal peptide (responsible for translocation to the ER); a prosegment of 40-50 amino acid residues (involved in the correct folding, stabilisation and sorting of the enzyme); and a mature enzyme possessing two catalytic sequence motifs. The two catalytic aspartate residues in plant aspartic proteases are contained within Asp-Thr-Gly and Asp-Ser-Gly motifs. With a few exceptions, the majority of plant aspartic protease identified so far are synthesized with a prepro-domain and subsequently converted to mature two-chain enzymes. Proteolytic processing of plant aspartic proteases starts with removal of the signal sequence upon translocation to the ER lumen. The following conversion steps include cleavage of the prosegment and total or partial removal of the internal PSI domain to produce mature two-chain forms of the enzymes. In the mature two-chain form both polypeptide chains are held together by hydrophobic interactions and hydrogen bonds (see Simões and Faro (2004)3).
Plant Specific Insert (PSI)
Many plant aspartic proteases differ from their mammalian and microbial counterparts by the presence of a plant-specific insert (PSI) which is cleaved out during protein maturation to give rise to the mature, two-chain enzyme. The PSI typically has about 104 amino acids. In phytepsin, from barley, removal of the PSI led to secretion of the mutated phytepsin when expressed in Tobacco protoplasts, whilst retaining enzymatic activity4. The presence of PSI was shown to be at least necessary for vacuolar sorting4. The PSI of Cardosin A has been shown to be an inducer of vesicle leakage18.
Vacuolar Sorting
The final destination of a protein after synthesis is a highly complex and regulated process and is usually dependent on the presence of specific targeting information (e.g. sorting signals, post-translational modifications) which is specifically recognized by receptors that target nascent proteins to their final localizations in the cell1.
One of the most complex biosynthetic routes is the secretory pathway. In a very simplified way, this system comprises several membrane-bound subcellular compartments and proteins are exchanged between these compartments by vesicle trafficking. Proteins resident in the endoplasmic reticulum (ER), Golgi apparatus, vacuoles or plasma membrane/extracellular matrix have to enter this endomembrane system and some of them undergo processing and post-translational modifications along their passage through the ER and Golgi network. Targeting to ER is determined cotranslationally by the presence of a signal peptide at the N-terminus of a nascent protein1. Although recent evidence indicates that the system may be more complex than first expected2, it is still generally accepted that proteins are actively sorted to vacuoles, meaning that they contain specific vacuolar sorting signals (VSS's).
Different types of vacuolar sorting signals (VSS's) have been identified1,3. Even though no consensus sequence has been yet defined for these signals they are currently divided into three categories: sequence-specific VSS (ssVSS's) which comprise N-terminal propeptides (e.g. sporamin) or internal sequences (e.g. ricin); C-terminal propeptides (CTPP's) (e.g. lectin and chitinase) and physical structure VSS (psVSS's) [e.g. plant specific Insert (PSI) of phytepsin]3. Given the number of soluble vacuolar proteins that lack these types of VSS's, it is expected that novel motifs for vacuolar sorting are yet to be identified.
The ability to manipulate protein sorting is particularly important if considering high value-protein expression in heterologous or homologous systems. Specifically sorting a selected protein to storage vacuoles may be highly advantageous for accumulation of large quantities of recombinant proteins and, thereby, increase the food value of a plant. Conversely, redirecting a native vacuolar protein for secretion may be particularly useful considering, for example, expression in heterologous systems like yeasts where protein secretion into the media greatly facilitates recombinant protein handling and purification.
The relevance of these vacuolar sorting signals in various applications is confirmed by different issued patents: US69723504, US73686285, US53607266 and US60546377, where the last two describe the VSS's of lectin and chitinase, respectively.
Typical aspartic proteases are widely distributed in plants and have been purified from a variety of tissues. In general, these enzymes share high levels of amino acid sequence identity (over 60%) and the majority of them accumulate inside plant vacuoles. However, there are exceptions to this intracellular localization and several plant aspartic proteases were shown to be extracellular8.
Plant aspartic proteases have been used in cheese manufacture for many years. Indeed, this is amongst the earliest application of enzymes in food processing, dating back to approximately 6000 BC (see Fox and McSweeney 1999 cited in Claverie-Martin and Vega-Hernandez (2007)19). Plant extracts, including dried flowers, have been added to milk to act as a coagulant. Although efforts have been made to produce recombinant plant aspartic proteases, these are not yet commercially available due to the large volumes of culture or high number of culture steps to obtain a significant amount of recombinant protein.